Genome-wide target analysis of Shh-activated transcription network in limb bud

我们的长期目标是揭示将基因调控和表达的早期模式与结构的最终实现联系起来的步骤，作为信号网络如何协调复杂组织形成的范例。为了实现这一目标，我们正在开发几种结合遗传学、基因组学和蛋白质组学的方法来研究在肢体发育过程中运作的转录因子和调控级联，最终目的是阐明早期诱导前后（AP）模式与不同指骨（指骨数量、长度和形状不同）形态发生之间的调控层次。了解转录因子如何在正常发育过程中协调生长和形态发生，将促进我们对如何治疗当这些调节成分突变或异常表达时出现的遗传疾病和癌症的理解。Hoxd基因如何指导手指身份特征（如关节数量、形状、大小），Hoxd与Gli3靶点之间的关系是什么？了解这种三维结构是如何形成的，对于理解器官发生是如何实现的通常是相关的。最近报道了一些5Hoxd的体内靶点（Shh增强子，Hand2启动子），尽管没有很好地表征。定义5Hoxd函数的时间窗对于选择肢体阶段进行ChIP分析非常重要。我们在不同时间去除Hoxd基因功能的遗传结果表明，这些基因在趾发育的相对较晚阶段调控趾形态和关节形成，此时趾的前体射线已经开始出现。我们已经开发了几种针对ChIP的抗体，并与Steve Vokes（美国德克萨斯州，奥斯汀）合作，成功地设计了一种带有抗标签的ChIP表位标记的Hoxd13条件转基因等位基因。这种可诱导的转基因还将促进靶标的生物学验证，以及我们已经在实验室中拥有的条件敲除等位基因。结果将与Vokes博士实验室的预期结果相关联，Vokes博士也在分析Gli3靶点，并为我们提供了一个表位标记的Gli3表达小鼠系。确定Hoxd和Gli3靶点将有助于深入了解共调控基因和Gli3-Hoxd的作用，并阐明Hoxd基因在肢体形态发生中的后期效应。肢体中由Hoxd和Gli3调控的转录网络也将被分析与sh通路靶标的关系，sh通路靶标形成两种不同的类别，需要短暂或持续的信号传导才能稳定激活。通过这种方式，我们希望揭示导致具有不同数量的节段和关节的定义趾形态形成的调控级联。Gli和Hox基因也在某些癌症中异常共表达，并可能导致其发病机制，这些研究也将阐明它们在这些情况下可能发挥的作用。

Our long term goal is to unravel the steps linking early patterns of gene regulation and expression with the ultimate realization of structure to serve as a paradigm for how signaling networks orchestrate the formation of a complex tissue. To accomplish this, we are developing several combined genetic and genomic, and proteomic approaches to study transcription factors and regulatory cascades operating during limb development with the ultimate aim of elucidating the regulatory hierarchy between early induction of antero-posterior (AP) pattern and the morphogenesis of distinct digits (with different numbers, lengths and shapes of phalanges). Learning how transcription factors orchestrate growth and morphogenesis during normal development will advance our understanding of how to treat genetic diseases and cancers that arise when such regulatory components are either mutated or expressed abnormally. How do Hoxd genes instruct features of digit identity (such as numbers of joints, shape, size) and what is the relation between Hoxd and Gli3 targets? Learning how this 3-dimensional structure forms will be generally relevant for understanding how organogenesis is achieved. A few 5Hoxd in vivo targets have been reported recently (Shh enhancer, Hand2 promoter), albeit not well-characterized. Defining time windows for 5Hoxd functions will be important in choice of limb stages for ChIP analysis. Our genetic results examining effects of removing Hoxd gene function at different times indicates that these genes regulate digit morphology and joint formation at relatively late stages of digit development, when precursor rays for digits have begun to appear. We have developed several antibodies for ChIP, and we have successfully engineered an epitope-tagged Hoxd13 conditional transgene allele for ChIP with anti-tag in collaboration with Steve Vokes (U. Texas, Austin). This inducible transgene will also facilitate biological validation of targets, along with a conditonal knock-out allele that we already have in the lab. Results will be correlated together with anticipated results from Dr. Vokes lab, who is also analyzing Gli3 targets, and who has made an epitope-tagged Gli3 expressing mouse line available to us. Identifying Hoxd and Gli3 targets will provide insight into co-regulated genes and Gli3-Hoxd roles as well as illuminating late effectors of Hoxd genes in limb morphogenesis. The transcriptional network regulated by Hoxd and Gli3 in the limb will also be analyzed in relation to Shh-pathway targets that form two distinct classes, requiring either transient or sustained signaling for their stable activation. In this manner, we hope to uncover the regulatory cascade leading to formation of defined digit morphologies with distinct numbers of segments and joints. Gli and Hox genes are also aberrantly co-expressed in some cancers and may contribute to their pathogenesis, and these studies will also shed light on their possible roles in these contexts.